Insect development from larva to adult depends on the repeated shedding of old cuticle via a highly stereotyped behavior called ecdysis. We recently identified a novel, segmentally distributed endocrine system of epitracheal glands (EG) in Manduca sexta that produce "ecdysis- triggering hormone" (ETH). Our preliminary data suggests that the brain peptide eclosion hormone, formerly thought to act on the nervous system, actually triggers ETH release from the EG. Released ETH provides an immediate chemical signal for ecdysis through its direct action on the central nervous system. Additional peptides associated with ETH (ETH-APs) and FMRFamide-like peptides (FLP) also are produced by the EG during the intermolt period and probably are required for the full expression of ecdysis behavior. The discovery of the epitracheal glands and ETH requires a re-definition of the endocrine basis of ecdysis in insects. The objectives of the proposal are to elucidate the regulatory mechanisms controlling the release of EG hormones and to define their functional roles in the orchestration of ecdysis, a complex behavioral process that is critical in the life history of insects. The first objective will be to isolate and obtain amino acid sequences for ETH-like peptides and FLPs. These sequences will be used for production of specific antisera and isolation of their cDNA precursors and genes. The second objective will be elucidate the mechanisms governing expression of release of ETHs and FLPs from the EG. The roles of ecdysteroids and juvenile hormone in regulation of ETH and FLP expression in the EG as well as the sensitivity of EGs to eclosion hormone and/or other potential releasing factors will be examined. The signal transduction steps involved in eclosion hormone-induced release of ETH and FLP from the EGs will be evaluated. The third objective will be to evaluate the signaling pathways underlying ETH action on the nervous system and to identify the ETH receptor. The long range goals of this proposal are to describe the molecular and physiological bases for ecdysis behavior in insects. The simplicity of the EG system, composed of glands containing only 1-3 cells, provides an excellent model for examination of endocrine function at the cellular, developmental, and molecular levels. Since ecdysis is crucial for successful development of insects and other arthropods, understanding the regulatory mechanisms which underlie these precisely timed and synchronized processes may help in the biological control of pest insects and possibly other arthropods.